Scaffolds fabricated from electrospun decellularized extracellular matrix

ABSTRACT

A scaffold comprising electrospun decellularized ECM of an organ, wherein the decellularized ECM has a similar protein composition to native ECM of the organ, Methods of generating same are also disclosed as well as uses of same.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates the use ofelectrospun decellularized extracellular matrix for fabricatingscaffolds.

Injured organs or tissues can be replaced via whole organtransplantation. However, major obstacles limit the surgical proceduresused for this purpose, including shortage of donors and the need to useimmunosuppressive drugs to prevent rejection of the implanted organ.Tissue engineering has emerged as a promising approach to improve orrestore the function or shape of a damaged tissue or organ byimplantation of polymeric scaffolds, functional cells, or theircombination in cell seeded scaffolds. The success of a scaffold fortissue engineering depends on the manufacturing design, the materialchosen, and on the accurate understanding of the scaffold's desiredcharacteristics.

A variety of scaffolds were designed and studied in the last two decadesfor the regeneration of tissues in various organs, using either naturalmaterials or synthetic ones. Moreover, tissue engineering is becoming animportant research tool in biology and biomedical research, providingvarious possibilities for 3D cultures, and thus bridging the gap between2D cultures and animal models.

Many scaffolds currently developed for tissue engineering are made ofsynthetic materials, such as PLEA, PMMA and PCL, whose main advantage isthat they can be custom-tailored depending on their specificapplications. They can be easily designed to match specific propertiessuch as degradability, density and mechanical strength. Their majordrawbacks, however, are limited biocompatibility and the absence of anybiological activity.

Another approach for tissue engineering is the use of natural polymerssuch as alginate, collagen and chitosan. These materials arebiologically active and typically promote excellent cell adhesion andgrowth. They are also biodegradable and allow host cells to eventuallyreplace them with their own extracellular matrix. However, scaffoldsfrom biological materials are difficult to control, or finely tune fordesired properties. They lack reproducibility and generally have poormechanical properties, hick limits their use.

In recent years, the use of whole decellularized extracellular matrix(ECM) was suggested as the ultimate biomaterial for tissue engineering,as it is the closest mimic to natural cell surroundings, it isbioactive, biodegradable and biocompatible. Whole decellularized ECM hasbeen either used “as-is” or dissolved and refabricated as a gel.However, while this top-down approach provides a more accuratebiological environment, it still suffers the drawbacks of naturalmaterials.

In view of these drawbacks, newly developed technologies were employedto to produce scaffolds of better controlled properties. One suchtechnology is electrospinning, which provide a bottom-up approach wherethe fibers are spun into a matte in an organized, homogeneous manner.Nonetheless, electrospinning of biological polymers is not easy sincethey tend to present the proper degree of viscosity, but lackvisco-elasticity, which is required for the electrospinning. Thus, thesesolutions of natural, biological polymers usually need to be combinedwith synthetic polymers in order to enhance the visco-elasticity forelectrospinning.

U.S. Patent Application No. 20120156250 teaches soluble decellularizedECM.

International Patent Application WO2006/138718 teaches electrospun ECMfor generating scaffolds. The ECM is not derived from decellularizedtissue.

Additional background art includes Gibson et al., BioMed ResearchInternational, 2014, Article ID 469120 and Francis NIP et al., 2012. JBiomed Mater Res Part A 2012:100A:1716-1724.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a scaffold comprising:

(a) homogenizing decellularized extracellular matrix (ECM) in an organicsolvent to generate a homogenate of decellularized ECM;

(b) electrospinning the homogenate onto a solid surface therebygenerating the scaffold.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a scaffold comprising:

(a) dissolving decellularized extracellular matrix (ECM) in an organicsolvent to generate a solution of decellularized ECM, wherein thedecellularized ECM is derived from an organ selected from the groupconsisting of heart and pancreas; and

(b) electrospinning the solution onto a solid surface thereby generatingthe scaffold.

According to an aspect of some embodiments of the present inventionthere is provided a scaffold generated according to the method describedherein.

According to an aspect of some embodiments of the present inventionthere is provided a scaffold comprising electrospun decellularized ECMof an organ, wherein the to decellularized ECM has a similar proteincomposition to native ECM of the organ.

According to an aspect of some embodiments of the present inventionthere is provided a scaffold comprising electrospun decellularized ECM,wherein the decellularized ECM is derived from an organ selected fromthe group consisting of heart and pancreas.

According to an aspect of some embodiments of the present inventionthere is provided a composition of matter comprising the scaffolddescribed herein and cells seeded on the scaffold.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a medical condition which maybenefit from cell transplantation in a subject in need thereof,comprising transplanting the scaffold described herein into the subject,thereby treating the medical condition.

According to some embodiments of the invention, the method furthercomprises decellularizing a tissue of a subject prior to generate thedecellularized ECM prior to step (a).

According to some embodiments of the invention, the method furthercomprises contacting the solution of decellularized. ECM with a polymerso as to increase the viscoelasticity of the solution following step (a)and prior to step (b).

According to some embodiments of the invention, the dissolving iseffected by homogenization to generate a homogenate of decellularizedECM.

According to some embodiments of the invention, the method furthercomprises filtering the homogenate of decellularized ECM prior to theelectrospinning.

According to some embodiments of the invention, the organic solvent isselected from the group consisting of acetone, N,N-dimethylformamide(DMF), diethylformamide, chloroform, methylethylketone, acetic acid,formic acid, ethanol, 1,1,1,3,3,3-hexa fluoro-2-propanol (HFIP),tetrafluoroethanol, dichloromethane (DCM), tetrahydrofuran (THF),trifluoroacetic acid (TFA), camphorsulfonic acid, dimethyl acetamide,isopropyl alcohol (IPA) and mixtures thereof.

According to some embodiments of the invention, the organic solvent isHFIP.

According to some embodiments of the invention, the polymer is abiocompatible polymer.

According to some embodiments of the invention, the polymer is ahydrophilic polymer.

According to some embodiments of the invention, the polymer is asynthetic polymer.

According to some embodiments of the invention, the synthetic polymer isselected from the group consisting of poly(D,L-lactide) poly(urethanes),poly(siloxanes), poly(silicones), poly(ethylene), poly(vinylpyrrolidone), poly(2-hydroxy ethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), polyvinyl alcohol) (PVA), polyacrylic acid), poly(vinyl acetate), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLGA),nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol)(EVOH), polycaprolactone, poly(vinyl acetate), polyvinylhydroxide,poly(ethylene oxide) (PEO), polyorthoesters and mixtures thereof.

According to some embodiments of the invention, the synthetic polymer isPEO.

According to some embodiments of the invention, the amount of the PEO inthe solution is between 0.05-1% mass.

According to some embodiments of the invention, the decellularized ECMis derived from an organ selected from the group consisting of heart andpancreas.

According to some embodiments of the invention, the decellularized ECMis derived from porcine tissue.

According to some embodiments of the invention, the method furthercomprises removing the polymer following the electrospinning.

According to some embodiments of the invention, the decellularized ECMhas a similar protein composition to native ECM of the organ.

According to some embodiments of the invention, the organ is a heart ora pancreas.

According to some embodiments of the invention, the organ is a humanorgan or a porcine organ.

According to some embodiments of the invention, the decellularized ECMcomprises collagen type I and collagen type III.

According to some embodiments of the invention, the decellularized ECMis devoid of collagen type VI.

According to some embodiments of the invention, the diameter of fibersof the scaffold are between 100-2000 nm.

According to some embodiments of e invention, the diameter of fibers ofthe scaffold are between 300 to 1500 nm.

According to some embodiments of the invention, the scaffold, whenhydrated has fibers of a similar organization to native ECM of theorgan.

According to some embodiments of the invention, the scaffold is devoidof a synthetic polymer.

According to some embodiments of the invention, the scaffold has beenpre-seeded with cells.

According to some embodiments of the invention, the medical condition isa cardiac disease.

According to some embodiments of the invention, the medical condition isDiabetes.

According to some embodiments of the invention, the scaffold is for usein treating a medical condition which may benefit from celltransplantation.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and-'or materials are described below. In case of conflict, thepatent specification, including definitions, will control, in addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-E are photographs of porcine ECM electrospun in: acidifiedwater and combined with PE( )to a mass ratio of (a) 7:1, (h) 8:1, and(c) 14:1. Electrospinning of pcECM dissolved in HFIP (d) without and (e)with 0.1 mass % PEO. Scale bar 100 μm.

FIGS. 2A-D are HR-SEM images of porcine cardiac ECM (pcECM) fibers at(A) 200 X, (B) 1 kX, (C) 5 kX, and (D) 10 kX.

FIGS. 3A-B are HR-SEM images of porcine pancreas ECM (ppECM) fibers.Scale bars 200 μm.

FIG. 4 is a graph illustrating the protein content of pcECM electrospunfibers.

FIG. 5 is a graph illustrating fiber diameter distribution.

FIG. 6 is a graph illustrating pore size distribution in the electrospunpcECM scaffold.

FIGS. 7A-B are photographs of initial wetting/contact angle of theelectrospun pcECM scaffold (A), and the wetting/contact angle after 5min (13).

FIG. 8 is a photograph of initial wetting/contact angle of theelectrospun ppECM scaffold.

FIGS. 9A-B are a graph illustrating the Fourier transform infraredspectroscopy (MR) spectra of the electrospun pcECM scaffold compared todecellularized pcECM (A), and the secondary structure of scaffoldproteins determined by Fourier deconvolution of their amid I band (B).

FIG. 10 is a graph illustrating the thermal gravimetric analysis (TGA)curves representing the degradation behavior over temperature of theelectrospun pcECM scaffold and decellularized pcECM.

FIGS. 11A-L are SEM images of decellularized pcECM (A,B), andelectrospun pcECM scaffolds wetted at different temperatures; scaffoldwetted at 37° C. for 1 hr and then at 37° C. overnight (C,D) scaffoldwetted at 24° C. for 1 hr and then at 24° C. overnight (E,F); scaffoldwetted at 4° C. for 1 hr and then at 4° C. overnight (G,H); scaffoldwetted at 24° C. for 1 hr and then at 37° C. overnight (I,J); scaffoldwetted at 4° C. for 1 hr and then at 37° C. overnight (K,L).

FIGS. 12A-D are graphs illustrating the mechanical properties of theelectrospun pcECM scaffold compared to native tissue in physiologicalsolution, visualized in terms of a stress-strain curve (A), maximumstress (B), strain at maximum stress (C), and Young's Modulus (D).

FIG. 13 is a graph illustrating the viability of hMSCs on pcECMelectrospun fibrous scaffolds after 24 h and 1 to 4 weeks.

FIGS. 14A-D are SEM images of hMSCs seeded on electrospun pcECM scaffoldafter 4 weeks in culture.

FIGS. 15A-C are images of hMSCs on pcECM electrospun fibrous scaffolds.Light sheet fluorescent microscopy (LSFM) images at 1 day (A) and 4weeks (B) using Hoechst 33258 (DNA-blue), while observing the ECMautofluorescence (red and green). Hematoxylin and eosin (H&E) stainingat 4 weeks post seeding (C).

FIGS. 16A-D are graphs illustrating the relative expressions of ECMremodeling genes by hMSCs grown on the electrospun pcECM scaffoldcompared to native pcECM. Collagen I (A) Collagen III (B) Tissueinhibitor of metalloproteinases type 1 (TIMP1) (C) Matrixmetalloproteinase-2 (MMP2) (D).

FIG. 17 is a LSFM image of electrospun pcECM scaffold seeded with humaninduced pluripotent stem cells (hiPSCs) at 3 weeks post seeding.

FIGS. 18A-D are images of hiPSCs cultured on pcECM electrospun fibrousscaffolds. SEM images (A-C) and H&E staining (D) at 3 weeks postseeding.

FIGS. 19A-C are images of cardiomyocytes cultured on pcECM electrospunfibrous scaffolds. SEM image (A) LSFM image (B) and H&E staining (C) at3 weeks post seeding.

FIGS. 20A-C are images of cardiomyocytes cultured on pcECM electrospunfibrous scaffolds for 3 weeks, stained with Hoechst 33258 (DNA-blue) andantibodies for Connexin-43 (q), sarcomeric α-actinin-SAA (r), andcardiac troponin 1 (cTn1, s) cardiac markers (green).

FIG. 21 is a confocal line scan images showing changes in intracellularCa²⁺ in a Fluo-4 loaded neonatal cardiomyocytes seeded electrospun(ES)-pcECM scaffold 2 weeks post seeding. Whole cell Ca²⁺ transient inthree different induced pacing frequencies are shown.

FIG. 22 is a LSFM image of electrospun pcECM scaffold seeded with hiPSCsthat were differentiated into cardiomyocytes (hiPSC-CM) at 3 weeks postseeding. green: phalloidin-FITC (Actin), blue: Hoechst (DNA).

FIG. 23 is a graph illustrating the viability of hiPSC-CMs on pcECMelectrospun scaffolds after 1, 7 and 14 days.

FIG. 24 is a confocal line scan images showing changes in intracellularCa²⁺ in a Fluo-4 loaded hiPSC-CM seeded electrospun pcECM scaffold 14days post seeding. Whole cell Ca²⁺ transient in 1Hz induced pacingfrequency are shown.

FIGS. 25A-C are graphs illustrating in vitro immunogenicity studies ofpcECM electrospun fibrous scaffolds. Pieces of electrospun pcECMscaffold were used to stimulate RAW macrophages, LPS stimulation was apositive control, and PLGA and non-stimulated cells were negativecontrols. The level of (A) secreted NO and excreted pro-inflammatorycytokines TNFα and (C) IL1β were evaluated.

FIGS. 26A-B are graphs illustrating pro-inflammatory cytokine expressionof (A) TNFα and (B) IL1β in inguinal lymph nodes of mice that received asubcutaneous implanted electrospun fibrous scaffold from PLGA (dots) andECM (stripes).

FIGS. 27A-I are graphs illustrating complete blood counts of micefollowing subcutaneous implantation of electrospun PLGA and electrospunpcECM scaffolds. Number of white blood cells (WBC, A), and red bloodcells (RBC, B), hematocrit volume (C), hemoglobin concentration (D),mean corpuscular volume (MCV, E) mean corpuscular hemoglobin (MCH, F),mean corpuscular hemoglobin concentration (MCHC, G) number ofneutrophils (H), and lymphocytes (I), all plotted over a period of fourweeks following implantation. Dashed lines represent ranges of normalblood values for C57 black mice (Charles River Laboratories).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to the useof electrospun decellularized extracellular matrix for fabricatingscaffolds.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Scaffolds developed for tissue repair and regenerative therapy requireappropriate structure and functional characteristics native to thetissue or organ under consideration. Native ECM is a promising candidatefor this purpose, as each tissue is comprised of a unique combinationand 3D structure of macromolecules that provides cells with thenecessary cues and mechanical support.

In recent years, the use of whole decellularized extracellular matrix(ECM) was suggested as the ultimate biomaterial for tissue engineering,as it is the closest mimic to natural cell surroundings, it isbioactive, biodegradable and biocompatible.

However, scaffolds from decellularized ECM are difficult to control, orfinely tune for desired properties. They lack reproducibility andgenerally have poor mechanical properties, which limits their use.

In order to overcome these limitations, the present inventors nowpropose electrospinning decellularized ECM for fabrication of biologicalscaffolds. This would not only provide cells with the naturalenvironment attributed to the ECM, but also provide control over thestructure of the fibrous ECM network, allowing the design of scaffoldswith specific properties such as degradability, density and mechanicalstrength.

Whilst reducing the present invention to practice, the present inventorsgenerated scaffolds which were fabricated from ECM isolated from asingle organ—pancreas or heart.

As is illustrated herein under and in the examples section whichfollows, the present inventors show that the cells seeded on thescaffolds had a polypeptide composition similar to the native ECM of theorgan from which it was derived—Tables 1-2. The average fiber diameterof the scaffold ranged from 300 to 1500 nm. Furthermore, upon hydrationfiber organization and diameter became similar to that of native ECM ofthe organ from which it was derived (FIGS. 11A-B). The inventors furtherdemonstrate that cells seeded on the scaffolds were capable of survivingfor at least four weeks (FIGS. 13, 17, 19 and 23). Scaffolds fabricatedaccording to the disclosed methods were shown to be non-immunogenic(FIGS. 25-27).

Thus, according to one aspect of the present invention, there isprovided a method of generating a scaffold comprising:

(a) dissolving decellularized extracellular matrix (ECM) of an organ inan organic solvent to generate a solution of decellularized ECM; and

(b) electrospinning the solution onto a solid surface thereby generatingthe scaffold.

As used herein the phrase “decellularized ECM” refers to theextracellular matrix which supports tissue organization (e.g., a naturaltissue) and underwent a decellularization process (i.e., a removal ofall cells from the organ) and is thus completely devoid of any cellularcomponents.

The decellularized ECM typically comprises a plurality of polypeptides(e.g. collagens). For example, the decellularized ECM comprises collagenalpha-2(I), collagen alpha-1(III) and collagen alpha-1(I). Additionally,the decellularized ECM may also comprise collagen alpha-2(IV), collagenalpha-1(V) and collagen alpha-1(II) (or fragments thereof) Preferably,the amount of collagen alpha-2(I), collagen alpha-1(III) and collagenalpha-1(I) is greater in the decellularized ECM of this aspect of thepresent invention than collagen alpha-2(IV), collagen alpha-1(V) andcollagen alpha-1(II).

Additionally, the decellularized ECM may also comprise smaller amountsof collagen alpha-2(VI), collagen alpha-3(VI) and collagen alpha-1(VI)or collagen alpha-1(IV) (or fragments thereof). Preferably, the amountof collagen alpha-2(I), collagen alpha-1(III) and collagen alpha-1(I) isgreater in the decellularized ECM of this aspect of the presentinvention than collagen alpha-2(VI), collagen alpha-3(VI) and collagenalpha-1(VI) or collagen alpha-1(IV).

The phrase “completely devoid of any cellular components” as used hereinrefers to being more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, (e.g., 100%) devoid of the cellular components present in thenatural (e.g., native) tissue. As used herein, the phrase “cellularcomponents” refers to cell membrane components or intracellularcomponents which make up the cell. Examples of cell components includecell structures es., organelles) or molecules comprised in same.Examples of such include, but are not limited to, cell nuclei, nucleicacids, residual nucleic acids (e.g., fragmented nucleic acid sequences),cell membranes and/or residual cell membranes (e.g., fragmentedmembranes) which are present in cells of the tissue. It will beappreciated that due to the removal of all cellular components from thetissue, such a decellularized matrix cannot induce an immunologicalresponse when implanted in a subject.

The phrase “extracellular matrix (ECM)” as used herein, refers to acomplex network of materials produced and secreted by the cells of thetissue into the to surrounding extracellular space and/or medium andwhich typically together with the cells of the tissue impart the tissueits mechanical and structural properties. Generally, the ECM includesfibrous elements (particularly collagen, elastin, or reticulin), celladhesion polypeptides (e.g., fibronectin, laminin and adhesiveglycoproteins), and space-filling molecules [usually glycosaminoglycans(GAG), proteoglycans].

According to embodiments of the present invention the decellularized ECMis derived from cardiac or pancreatic tissue.

Other tissues contemplated by the present inventors include braintissue, bone tissue, muscle, liver, kidney, blood vessel, lung andplacenta.

In other embodiments, the decellularized ECM is not derived from fattissue.

In still other embodiments, the decellularized ECM is not MATRIGEL orderived from isolated basement membranes.

The ECM may be derived from an autologous or non-autologous subject(e.g., from allogeneic or even xenogeneic tissue, due tonon-immunogenicity of the resultant decellularized matrix). The tissueis removed from the subject [e.g., an animal, preferably a mammal, suchas a pig, rat, monkey or chimpanzee, or alternatively, a deceased humanbeing (shortly after death)] and washed e.g. in a sterile salinesolution (0.9% NaCl, pH=7.4) or phosphate buffered saline (PBS), whichcan be supplemented with antibiotics such as Penicillin/Streptomycin 250units/ml. Although whole tissues can be used, for several applications,segments of tissues may be cut e.g. sliced. Such tissue segments can beof various dimensions, depending on the original tissue used and thedesired application.

To remove the vasculature surrounding and feeding the tissue, the tissuemay be washed at room temperature by agitation in large amounts (e.g.,50 ml per each gram of tissue segment) of EDTA solution (0.5-10 mM,pH-7.4).

Next, the tissue is subjected to a hypertonic or hypotonic buffer tothereby obtain increased intercellular space within the tissue.

The hypertonic buffer used by the present invention can be any buffer orsolution with a concentration of solutes that is higher than thatpresent in the cytoplasm and/or the intercellular liquid within thetissue [e.g., a concentration of NaCl which is higher than 0.9% (w/v)].Due to osmosis, incubation of the tissue with the hypertonic bufferresults in increased intercellular space within the tissue.

According to another embodiment, peracetic acid is used to decellularizethe tissue.

Preferably, the hypertonic buffer used by the method according to thisaspect of the present invention includes sodium chloride (NaCl) at aconcentration which is higher than 0.9% (w/v), preferably, higher than1% (w/v), preferably, in the range of 1-1.2% (w/v), e.g., 1.1% (w/v).

Preferably, the hypotonic buffer used by the method according to thisaspect of the present invention includes sodium chloride (NaCl) at aconcentration which is lower than 0.9% (w/v), lower than 0.8% (w/v),lower than 0.7% (w/v), preferably, in the range of 0.6-0.9 % (w/v),e.g., 0.7 % (w/v).

According to this aspect of the present invention, the tissue issubjected to the hypertonic or hypotonic buffer for a time periodleading to the biological effect, i.e., cell shrinkage which leads toincreased intercellular space within the tissue.

According to a particular embodiment, the tissue is contacted with ahypertonic buffer (e.g. 1.1% w/v) and subsequently contacted with ahypotonic buffer (e.g. 0.7% w/v). This procedure may be repeated for twoor more cycles.

Preferably, the hypotonic buffer used by the method according to thisaspect of the present invention includes sodium chloride (NaCl) at aconcentration which is lower than 0.9% (w/v), lower than 0.8% (w/v),lower than 0.7% (w/v), preferably, in the range of 0.6-0.9% (w/v), e.g.,0.7% w/v.

Following incubation with the hypertonic/hypotonic buffer, the tissue isfurther subjected to an enzymatic proteolytic digestion which digestsall cellular components within the tissue yet preserves the ECMcomponents (e.g., collagen and elastin) and thus results in a matrixwhich exhibits the mechanical and structural properties of the originaltissue ECM. It will be appreciated that measures are taken to preservethe ECM components while digesting the cellular components of thetissue. These measures are further described hereinbelow and include,for example, adjusting the concentration of the active ingredient (e.g.,trypsin) within the digestion solution as well as the incubation time.

Proteolytic digestion according to this aspect of the present inventioncan be effected using a variety of proteolytic enzymes. Non-limitingexamples of suitable proteolytic enzymes include trypsin and pancreatinwhich are available from various sources such as from Sigma (St Louis,Mo., USA). According to one preferred embodiment of this aspect of thepresent invention, proteolytic digestion is effected using trypsin.

Digestion with trypsin is preferably effected at a trypsin concentrationranging from 0.01-0.25% (w/v), more preferably, 0.02-0.2% (w/v), morepreferably, 0.05-0.1 (w/v), even more preferably, a trypsinconcentration of about 0.05% (w/v). For example, a trypsin solutioncontaining 0.05% trypsin (w/v; Sigma), 0.0% EDTA (w/v;) and antibiotics(Penicillin/Streptomycin 250 units/ml), pH=7.2] may be used toefficiently digest all cellular components of the tissue.

It will be appreciated that for efficient digestion of all cellularcomponents of the tissue, each of the tissue segments may be placed in aseparate vessel containing the digestion solution (e.g., a trypsinsolution as described hereinabove) in a ratio of 40 ml digestionsolution per each gram of tissue. Preferably, while in the digestionsolution, the tissue segments are slowly agitated (e.g., at about 150rpm) to enable complete penetration of the digestion solution to allcells of the tissue.

It should be noted that the concentration of the digestion solution andthe incubation time therein depend on the type of tissue being treatedand the size of tissue segments utilized and those of skilled in the artare capable of adjusting the conditions according to the desired sizeand type of tissue.

Preferably, the tissue segments are incubated for at least about 20hours, more preferably, at least about 24 hours. Preferably, thedigestion solution is replaced at least once such that the overallincubation time in the digestion solution is at least 40-48 hours.

Next, the cellular components are removed from the tissue. Removal ofthe digested components from the tissue can be effected using variouswash solutions, such as detergent solutions (e.g., ionic and non ionicdetergents such as SDS Triton X-100, Tween-20, Tween-80) which can beobtained from e.g., Sigma (St Louis, Mo., USA) or Biolab (Atarot,Israel, Merck Germany).

Preferably, the detergent solution used by the method according to thisaspect of the present invention includes TRITON-X-100 (available fromMerck). For efficient removal of all digested cellular components,TRITON-X-100 is provided at a concentration range of 0.05-2.5% (v/v),more preferably, at 0.05-2% (v/v), more preferably at 0.1-2% (v/v), evenmore preferably at a concentration of 1% (v/v).

Preferably, for optimized results, the detergent solution includes alsoammonium hydroxide, which together with the TRITON-X-100, assists inbreaking and dissolving cell nuclei, skeletal proteins, and membranes.

Preferably, ammonium hydroxide is provided at a concentration of0.05-1.5% (v/v), more preferably, at a concentration of 0.05-1% v/v),even more preferably, at a concentration of 0.1-1% (v/v) (e.g., 0.1%).

The concentrations of TRITON-X-100 and ammonium hydroxide in thedetergent solution may vary, depending on the type and size of tissuebeing treated and those of skills in the art are capable of adjustingsuch concentration according to the tissue used.

Incubation of the tissue (or tissue segments) with the detergentsolution can last from a few minutes to hours to even several days,depending on the type and size of tissue and the concentration of thedetergent solution used and those of skills in the art are capable ofadjusting such incubation periods. Preferably, incubation with thedetergent solution is effected for at least 24-72 hours. According toone embodiment, 2-4 cycles of incubation with the detergent solution areperformed until no foam is observed, such that the total incubation timemay be between about 150-200 hours.

Although as described hereinabove, incubation with the detergentsolution enables the removal of cell nuclei, proteins and membrane, themethod according to this aspect of the present invention optionallyincludes an additional step of removing nucleic acids (as well asresidual nucleic acids) from the tissue to thereby obtain a nucleic acidfree tissue. As used herein the phrase “nucleic acid-free tissue” refersto a tissue being more than 99% free of any nucleic acid or fragmentsthereof as determined using conventional methods (e.g.,spectrophotometry, electrophoresis). Such a step utilizes a DNasesolution (and optionally also an RNase solution). Suitable nucleasesinclude DNase and/or RNase [Sigma, Bet Haemek Israel, 20 μg/ml in Hankbalance salt solution (HBSS)].

The above described detergent solution is preferably removed bysubjecting the ECM to several washes in water or saline (e.g., at least10 washes of 30 minutes each, and 2-3 washes of 24 hours each), untilthere is no evident of detergent solution in the matrix.

Optionally, the decellularized ECM is then sterilized. Sterilization ofthe decellularized ECM may be effected using methods known in the art(e.g. 70% ethanol).

Typically, in order to carry out solubilization of the decellularizedECM, it is frozen (e.g. in liquid nitrogen), cut into small pieces (e.g.crumbled, crushed or ground) and then lyophilized.

The lyophilized decellularized ECM is then solubilized in an organicsolvent.

Exemplary organic solvents contemplated by the present inventorsinclude, but are not limited to acetone, N,N-dimethylformamide (DMF),diethylformamide, chloroform, methylethylketone, acetic acid, formicacid, ethanol, 1,1,1,3,3,3-hexa fluoro-2-propanol (HTIP),tetrafluoroethanol, dichloromethane (DCM), tetrahydrofuran (THF),trifluoroacetic acid (TFA), camphorsulfonic acid, dimethyl acetamide,isopropyl alcohol (IPA) and mixtures thereof.

Exemplary concentrations of decellularized ECM in the organic solventcontemplated by the present invention are between 0.005 g/mL to 0.5g/mL—for example about 0.05 g/mL.

According to a particular embodiment, the organic solvent is HFIP.

In order to aid in the solubilization process, following or concomitantwith the solubilization, the decellularized ECM may be homogenized.

Typically, the homogenization is effected in the presence of a rigidgrinding media which is preferably spherical or particulate in formhaving an average size less than about 10 mm (e.g. between 2-10 mm) and,more preferably between 2-7 mm. The selection of material for thegrinding media is not believed to be critical. Zirconium oxide, such as95% ZrO stabilized with magnesia, zirconium silicate, ceramic, stainlesssteel, titania, alumina, 95% ZrO stabilized with yttrium, glass grindingmedia, and polymeric grinding media are exemplary grinding materials.

The grinding media can comprise particles that are preferablysubstantially spherical in shape, e.g., beads_(;) consisting essentiallyof polymeric resin or other suitable material. Alternatively, thegrinding media can comprise a core having a coating of a polymeric resinadhered thereon.

The homogenization may be performed using a homogenizer e.g, a beadhomogenizer such as g a Precellys™ 24 bead. The homogenization should beeffected for a length of time until the solution appears homogeneous (at6000 rpm for 5 second intervals, for at least 6 intervals).

Optionally, to improve homogenization the homogenate may be sonicated.In one embodiment decellularized ECM derived from the pancreas issonicated (for example, for no more than three minutes) following thehomogenization step.

Following homogenization, the homogenate may be mixed for a suitablelength of time (e.g. 1 day, two days, three days or more) by placing ona rotator.

To remove any particulate matter, the homogenate may be filtered (e.g,using glass wool).

As mentioned, following the dissolution of the decellularized ECM_(;)the solution is then electrospun.

The present invention contemplates contacting the solution ofdecellularized. ECM with a polymer so as to increase the viscoelasticityof the solution prior to the electrospinning process.

In one embodiment, the polymer is a biocompatible polymer.

In another embodiment, the polymer is a hydrophilic polymer.

Preferably, the polymer is a synthetic polymer.

Exemplary synthetic polymers contemplated by the present inventioninclude, but are not limited to poly(D,L-lactide) (PLA),poly(urethanes), poly(siloxanes), poly(silicones), poly(ethylene),polyvinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), polyvinyl alcohol) (PVA),poly(acrylic acid), poly(vinyl acetate), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLEA),nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol)(EVOH), polycaprolactone, poly(vinyl acetate), polyvinylhydroxide,poly(ethylene oxide) (PEO), polyorthoesters and mixtures thereof.

According to a particular embodiment, the polymer is PEO.

The amount of polymer (e.g. PEO) in the solution is typically between0.05-1% mass, and more preferably between 0.05-0.5% mass—for exampleabout 0.1%.

As used herein, the term “electrospinning” refers to a technology whichproduces electrospun fibers (e.g. nanofibers and/or microfibers) from apolymer solution. During this process, the dissolved polymers are placedin a dispenser. An electrostatic field is employed to generate apositively charged jet from the dispenser to the collector. Thus, adispenser (e.g., a syringe with metallic needle) is typically connectedto a source of high voltage, preferably of positive polarity, while thecollector is grounded, thus forming an electrostatic field between thedispenser and the collector. Alternatively, the dispenser can begrounded while the collector is connected to a source of high voltage,preferably with negative polarity. As will be appreciated by oneordinarily skilled in the art, any of the above configurationsestablishes motion of positively charged jet from the dispenser to thecollector. Reverse polarity for establishing motions of a negativelycharged jet from the dispenser to the collector is also contemplated. Atthe critical voltage, the charge repulsion begins to overcome thesurface tension of the liquid drop. The charged jets depart from thedispenser and travel within the electrostatic field towards thecollector. Moving with high velocity in the inter-electrode space, thejet stretches and the solvent therein evaporates, thus forming fiberswhich are collected on the collector forming the electrospun scaffold.

Several parameters may affect the diameter of the fiber, these include,the size of the dispensing hole of the dispenser, the dispensing rate,the strength of the electrostatic field, the distance between thedispenser and/or the concentration of the polymer used for fabricatingthe electrospun fiber.

In one embodiment, the fibers are electrospun with a voltage of 1-20 kV,for example between 5-10 kV and more preferably between 8-9 kV. The holeof the dispenser may be between 20-30 gauge (e.g. a 23 gauge bluntneedle), and the distance from needle to collector may be between 1-20cm, more preferably between 6-10 cm, with a flow rate between 0.1-3ml/hr, more preferably between 0.5-1 ml/hr.

The electrospun fibers are collected on a solid surface such as a metalsurface or a polymeric surface. In one embodiment, the fibers arecollected on a metal surface coated with a polymer (for examplepolyethylene).

As mentioned, in some embodiments a polymer is added to the dissolveddecellularized ECM. The present invention contemplates removing thepolymer following the electrospinning, especially if the polymer is notbiocompatible. When the polymer is a hydrophilic polymer, it may beremoved simply by rinsing in an aqueous solution.

In one aspect of the present invention, the scaffolds compriseelectrospun decellularized ECM of an organ, wherein the decellularizedECM has a similar protein composition to native ECM of the organ.

As used herein, the term “scaffold” refers to a three dimensionalstructure comprising a biocompatible material that provides a surfacesuitable for adherence and proliferation of cells. A scaffold mayfurther provide mechanical stability and support.

The dimensions and shape of the scaffold will vary according to thedisease or injury being treated. It will be further appreciated that thedimensions of the scaffold will vary according to the size of thesubject.

Typically, the scaffolds of the present invention are porous. Theporosity of the scaffold may be controlled by altering the parametersused for electrospinning, as known to those skilled in the art. Theminimum pore size and degree of porosity is dictated by the need toprovide enough room for the cells and for nutrients to filter throughthe scaffold to the cells. The maximum pore size and porosity is limitedby the ability of the scaffold to maintain its mechanical stabilityafter seeding.

According to a preferred embodiment of this aspect of the presentinvention, the scaffold has an average pore diameter of about 10-40 μm.

According to this aspect the decellularized ECM may be derived from anytissue such as cardiac tissue, pancreatic tissue, blood vessel tissue,muscle tissue, liver tissue, kidney tissue, brain tissue, bone tissue,lung and placenta.

Preferably, the decellularized ECM is derived from pancreatic tissue orcardiac tissue.

When the decellularized ECM is derived from cardiac tissue,decellularized ECM maintains the protein composition of native cardiacECM, or at least has a similar protein composition to native cardiacECM. Thus, for example the majority of the collagen of thedecellularized ECM is collagen type I and type III, since that is themost abundant collagen in cardiac tissue.

In another embodiment, the decellularized ECM is devoid of collagen typeVI.

When the decellularized ECM is derived from pancreatic tissue,decellularized ECM maintains the protein composition of nativepancreatic ECM or at least has a similar protein composition to nativepancreatic ECM. Thus, for example the majority of the collagen of thedecellularized ECM are collagens type I and type III, since that is themost abundant collagen in pancreatic tissue.

In another aspect, the scaffold comprises electrospun decellularizedECM, wherein the decellularized ECM is derived from an organ selectedfrom the group consisting of heart and pancreas.

The scaffold comprises fibers of different thickness. When imaged usingimageJ analyses, thick fibers may have an average diameter between100-2000 nm, more preferably between 300-1500 nm (which corresponds totype I collagen fibers in native ECM) and thin fibers have an averagediameter of between 30-80 nm (which corresponds to type III collagenfibers in native ECM).

Thus, the present inventors contemplate that scaffolds generatedaccording to methods described herein are of a similar proteincomposition to native ECM, have a similar fiber diameter to native ECMand/or have a similar organization to native ECM when hydrated.

According to particular embodiments, the scaffolds are devoid of asynthetic polymer (do not comprise more than trace amounts of syntheticpolymer).

Therapeutic compounds or agents that modify cellular activity can alsobe incorporated (e.g. attached to, coated on, embedded or impregnated)into the scaffold material. Furthermore, the present inventorscontemplate embedding particles which release the therapeutic compoundsor agents into the scaffold. Campbell et al (US Patent Application No.20030125410) which is incorporated by reference as if fully set forth byreference herein, discloses methods for fabrication of 3D scaffolds forstem cell growth, the scaffolds having preformed gradients oftherapeutic compounds. The scaffold materials, according to Campbell etat, fall within the category of “bio-inks”, Such “bio-inks” are suitablefor use with the compositions and methods of the present invention.

Exemplary agents that may be incorporated into the scaffold of thepresent invention include, but are not limited to those that promotecell adhesion (e.g. fibronectin, integrins), cell colonization, cellproliferation, cell differentiation, anti-inflammatories, cellextravasation and/or cell migration. Thus, for example, the agent may bean amino acid, a small molecule chemical, a peptide, a polypeptide, aprotein, a DNA, an RNA, a lipid and/or a proteoglycan.

Proteins that may be incorporated into the scaffolds of the presentinvention include, but are not limited to extracellular matrix proteins,cell adhesion proteins, growth factors, cytokines, hormones, proteasesand protease substrates. Thus, exemplary proteins include vascularendothelial-derived growth factor (Vain, activin-A, retinoic acid,epidermal growth factor, bone morphogenetic protein, TGFβ, hepatocytegrowth factor, platelet-derived growth factor, TGFα, IGF-I and II,hematopoetic growth factors, heparin binding growth factor, peptidegrowth factors, erythropoietin, interleukins, tumor necrosis factors,interferons, colony stimulating factors, basic and acidic fibroblastgrowth factors, nerve growth factor (NGF) or muscle morphogenic factor(MMP). The particular growth factor employed should be appropriate tothe desired cell activity. The regulatory effects of a large family ofgrowth factors are well known to those skilled in the art.

The scaffolds of the invention may be seeded with cells, including forexample primary cells, cultured cells, single cell suspensions of cells,clusters of cells e.g. islets, cells which are comprised in tissuesand/or organs etc.

Cells can be seeded in the scaffold by static loading, or, morepreferably, by seeding in stirred flask bioreactors (scaffold istypically suspended from a solid support), in a rotating wall vessel, orusing direct perfusion of the cells in medium in a bioreactor. Highestcell density throughout the scaffold is achieved by the latter (directperfusion) technique.

The cells may be seeded directly onto the scaffold, or alternatively,the cells may be mixed with a gel which is then absorbed onto theinterior and exterior surfaces of the scaffold and which may fill someof the pores of the scaffold. Capillary forces will retain the gel onthe scaffold before hardening, or the gel may be allowed to harden onthe scaffold to become more self-supporting. Alternatively, the cellsmay be combined with a cell support substrate in the form of a geloptionally including extracellular matrix components. An exemplary gelis Matrigel™, from Becton-Dickinson. Matrigel™ is a solubilized basementmembrane matrix extracted from the EHS mouse tumor (Kleinman, H. K., etal, Biochem. 25:312, 1986). The primary components of the matrix arelaminin, collagen I, entactin, and heparan sulfate proteoglycan(perlecan) (Vukicevic, S., et al., Exp. Cell Res. 202:1, 1992).Matrigel™ also contains growth factors, matrix metalloproteinases (MMPs[collagenases]), and other proteinases (plasminogen activators [PAs])(Mackay, A. R., et al., BioTechniques 15:1048, 1993). The matrix alsoincludes several undefined compounds (Kleinman, H. K., et al., Biochem.25:312, 1986; McGuire, P. G. and Seeds, N. W., J. Cell. Biochem. 40:215,1989), but it does not contain any detectable levels of tissueinhibitors of metalloproteinases (TIMPs) (Mackay, A. R., et al.,BioTechniques 15:1048, 1993).

Alternatively, the gel may be growth-factor reduced Matrigel, producedby removing most of the growth factors from the gel (see Taub, et at.,Proc. Natl. Acad. Sci. USA (1990); 87 (10:4002-6). In anotherembodiment, the gel may be a collagen I gel, alginate or agar. Such agel may also include other extracellular matrix components, such asglycosaminoglycans, fibrin, fibronectin, proteoglycans, andglycoproteins. The gel may also include basement membrane componentssuch as collagen IV and laminin. Another gel contemplated by the presentinventors is an ECM gel—see for example Uriel et al., TissueEngineering: Part C. Volume 15, Number 3, 2009, Mary Ann Liebert, Inc.,DOI: 10.1089=ten.tec.2008.0309.

Enzymes such as proteinases and collagenases may be added to the gel, asmay cell response modifiers such as growth factors and chemotacticagents.

The cells may be derived from any organism including for examplemammalian cells, (e.g. human), plant cells, insect cells, algae cells,fungal cells (e.g. yeast cells), prokaryotic cells (e.g. bacterialcells).

According to a particular embodiment the cells comprise stem cells—e.g.adult stem cells such as mesenchymal stem cells or pluripotent stemcells such as embryonic stem cells or induced pluripotent stem cells.The stem cells may be modified so as to undergo ex vivo differentiation.

According to a particular embodiment, the cells are preferably intact(i.e. whole), and preferably viable, although it will be appreciatedthat pre-treatment of cells, such as generation of cell extracts ornon-intact cells are also contemplated by the present invention.

The cells may be fresh, frozen or preserved in any other way known inthe art cryopreserved).

According to another embodiment, the cells are derived from the pancreasor the heart.

The tissue from which the decellularized extracellular matrix isproduced may be selected (i.e. matched) according to the cells which areincorporated therein.

Thus, for example when the cells are derived from the pancreas—e.g.pancreatic beta cells (or modified so as to imitate pancreatic betacells), according to certain embodiments, the tissue from which thedecellularized extracellular matrix is produced is pancreatic tissue.

In a similar fashion, when the cells are derived from cardiactissue—e.g. cardiac myocardial cells (or modified so as to imitatecardiac myocardial cells), according to certain embodiments, the tissuefrom which the decellularized extracellular matrix is produced iscardiac myocardial tissue.

Typically, the cells secrete a factor (e.g. a polypeptide) that isuseful for the treatment of a disease.

Such factors include for example, hormones including but not limited toinsulin, thyroxine, growth hormone, testosterone, oestrogen,erythropoietin and aldosterone; enzymes, including but not limited tolysosomal enzyme such as glucocerebrosidase (GCD), acidsphingomyelinase, hexosaminidase, α-N-acetylgalactosaminidi se, acidlipase, α-galactosidase, α-L-iduronidase, iduronate sulfatase,α-mannosidase, sialidase, αfucosidase, G_(M1)-β-galctosidase, ceramidelactosidase, arylsulfatase A, β galactosidase and ceramidase; clottingfactors such as factor VIII.

According to a preferred embodiment, the cells secrete insulin.

As used herein, the term “insulin” refers to an insulin obtained bysynthesis or recombination, in which the peptide sequence is thesequence of human insulin, includes the allelic variations and thehomologs. The polypeptide sequence of the insulin may be modified toimprove the function of the insulin (e.g. long lasting).

According to one embodiment, the cells are naïve (non-geneticallymodified).

The present invention also contemplates use of cells which have beengenetically modified to express a recombinant protein. The recombinantprotein may be a therapeutic protein or may promote in vivo longevity(AM, adrenomedullin, Jun-Ichiro et al. Tissue Eng. 2006) or may promoteneurotransmitter release (e.g., such as by transfecting with tyrosinehydroxylase).

Examples of therapeutic, recombinant proteins that may be expressed inthe cells of the present invention include, but are not limited to anantibody, insulin, human growth hormone (rHGH), follicle stimulatinghormone, factor VIII, erythropoietin, Granulocyte colony-stimulatingfactor (G-CSF),alpha-glactosidase A, alpha-L-iduronidase (rhIDU;laronidase),N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase) Tissueplasminogen activator (TPA), Glucocerebrosidase, Interferon (IF)Interferon-beta-1a, Interferon beta-1b, Insulin-like growth factor 1(IGF-1), somatotropin (ST) and chymosin.

Other examples of exogenous polynucleotides which may be expressed inaccordance with the present teachings include, but are not limited to,polypeptides such as peptide hormones, antibodies or antibody fragments(e.g., Fab), enzymes and structural proteins or dsRNA,antisense/ribozyme transcripts which can be directed at specific targetsequences transcripts of tumor associated genes) to thereby downregulateactivity thereof and exert a therapeutic effect. Similarly, protectiveprotein antigens for vaccination (see, for example, Babiuk S et al JControl Release 2000;66:199-214) and enzymes such as fibrinolysin fortreatment of ischemic damage (U.S. Pat No. 5,078,995 to Hunter et al)may expressed in the cells for transdermal or transcutaneous delivery.The therapeutic protein can also be a prodrug.

Thus, according to another aspect of the present invention there isprovided a method of treating a medical condition (e.g., pathology,disease, syndrome, trauma) which may benefit from cell transplantationin a subject in need thereof comprising transplanting the scaffold ofthe present invention into the subject.

As used herein the term “treating” refers to inhibiting or arresting thedevelopment of a pathology and/or causing the reduction, remission, orregression of a pathology. Those of skill in the art will understandthat various methodologies and assays can be used to assess thedevelopment of a pathology, and similarly, various methodologies andassays may be used to assess the reduction, remission or regression of apathology. Preferably, the term “treating” refers to alleviating ordiminishing a symptom associated with a disease or trauma which maybenefit from cell transplantation. Preferably, treating cures, e.g.,substantially eliminates, the symptoms associated with the medicalcondition.

As used herein “a medical condition which may benefit from celltransplantation” refers to any medical condition which may be alleviatedby administration of the scaffold (cell-seeded, or non cell-seeded) ofthe present invention.

Examples of such medical conditions include, but are not limited to,stem cell deficiency, heart disease, neurodegenerative diseases,glaucoma neuropathy, Parkinson's disease, cancer, Schizophrenia,Alzheimer's disease, stroke, burns, loss of tissue, loss of blood,anemia, autoimmune disorders, diabetes, arthritis, graft vs. hostdisease (GvHD), neurodegenerative disorders, chronic pain, autoimmuneencephalomyelitis (EAE), systemic lupus erythematosus (SLE), rheumatoidarthritis, systemic sclerosis, Sjorgen's syndrome, multiple sclerosis(MS), Myasthenia Gravis (MG), Guillain-Barre Syndrome (GBS), Hashimoto'sThyroiditis (HT), Graves's Disease, Insulin Dependent Diabetes Melitus(IDDM) and Inflammatory Bowel Disease.

As mentioned, the method may be applied to repair cardiac tissue in ahuman subject having a cardiac disorder so as to thereby treat thedisorder. The method can also be applied to repair cardiac tissuesusceptible to be associated with future onset or development of acardiac disorder so as to thereby inhibit such onset or development.

The present invention can be advantageously used to treat disordersassociated with, for example, necrotic, apoptotic, damaged,dysfunctional or morphologically abnormal myocardium. Such disordersinclude, but are not limited to, ischemic heart disease, cardiacinfarction, rheumatic heart disease, endocarditis, autoimmune cardiacdisease, valvular heart disease, congenital heart disorders, cardiacrhythm disorders, impaired myocardial conductivity and cardiacinsufficiency.

According to one embodiment, the method according to this aspect of thepresent invention can be advantageously used to efficiently reverse,inhibit or prevent cardiac damage caused by ischemia resulting frommyocardial infarction.

According to another embodiment, the method according to this aspect ofthe present invention can be used to treat cardiac disorderscharacterized by abnormal cardiac rhythm, such as, for example, cardiacarrhythmia.

As used herein the phrase “cardiac arrhythmia” refers to any variationfrom the normal rhythm of the heart beat, including, but not limited to,sinus arrhythmia, premature heat, heart block, atrial fibrillation,atrial flutter, pulsus alternans and paroxysmal tachycardia.

According to another embodiment, the method according to this aspect ofthe present invention can be used to treat impaired cardiac functionresulting from tissue loss or dysfunction that occur at critical sitesin the electrical conduction system of the heart, that may lead toinefficient rhythm initiation or impulse conduction resulting inabnormalities in heart rate.

The term or phrase “transplantation”, “cell replacement”, “implantation”or “grafting” are used interchangeably herein and refer to theintroduction of the cells of the present invention to target tissue.

As used herein the term “subject” refers to any subject (e.g., mammal),preferably a human subject.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention, Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R, M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Willey & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunolog” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other procedures used in thecontext of the present invention are of the mechanical engineering andelectrospinning realm. Other general references are provided throughoutthis document. The procedures therein are believed to be well known inthe art and are provided for the convenience of the reader. All theinformation contained therein is incorporated herein by reference.

Example 1 Materials and Methods

Decellularization procedure: Porcine cardiac or pancreas ECM wasdecellularized and sterilized according to a previously publishedprotocol.²¹(Chaimov 2016, PMID: 27476611) Briefly, tissue of healthycommercial slaughter-weight pigs was isolated for the decellularizationprocedure. The procedure was comprised of two cycles with the followingstages: Alternating hyper/hypo tonic NaCl solutions; enzymatic treatmentusing trypsin; and detergent washes with Triton-X-100.

ECM dissolution: Decellularized, sterilized ECM was frozen in liquidnitrogen and crushed in a cryogenic tissue grinder (BioSpec,Bartlesville, Okla., USA), and subsequently placed on a lyophilizeruntil dry. Lyophillized ECM was dissolved in hexafluoroisopropanol(HFIP) to a concentration of 0.05 g/ml and homogenized using ZrO beadson a Precellys™ 24 bead homogenizer (Bertin Technologies, Rockville,Md., USA) at 6000 rpm until the solution appeared homogeneous,Pancreatic ECM was sonicated for up to 3 minutes following thehomogenization. Solutions were then filtered through glass fibers priorto electrospinning.

Electrospinning Conditions:

PEO was added only to solutions of porcine cardiac (pcECM) to obtain afinal solution of 0.1 mass % PEO. The porcine pancreatic ECM (ppECM) orpcECM/PEO solution was electrospun using a custom built electrospinningdevice. The fibers were electrospun with a voltage of 8-9 kV, andcollected on a thin film of polyethylene covering a rotating aluminumdisc with a diameter of 9 cm and a width of 11 mm. The capillary was a23 gauge blunt needle, and the distance from needle to collector was6-10 cm, and the flow rate was 0.5-1 ml/hr. Fibers were collected untila thick matte was observed. The matte was then peeled off the surfaceand placed in a dry environment. PEO was removed from pcECM matrices bywashing in a water-based solution, while the pcECM fibers remainedintact.

SEM: Samples were visualized using a FEG Zeiss Ultra Plus highresolution scanning electron microscope (HR-SEM, Zeiss, Jena, Germany),equipped with a Schotkky field emission electron gun to provide highlevel brightness that overcomes charging of uncoated non-conductivespecimens. The microscope is also equipped with a charge compensationsystem to further enhance this effect. Samples were mounted on aluminumstabs stubs using adhesive carbon double-sided tape, and images werecaptured at 1.3 kV accelerating voltage using a combination of thein-lens type and SE2 type detectors. The analysis of fiber diameters wasdetermined by taking an average of 20 measurements chosen at randomacross images take from 6 randomly selected areas of the scaffold thatwere at least a magnification of 5 kX. Digital images were analyzedevaluated using Image) software (version 1.49) and Porometric software(PhenomWorld).

Wetted scaffolds were visualized using a Phenotn ProX desktop SEM(PhenomWorld, Eindhoven, Netherlands), equipped with a temperaturecontrolled sample holder (Deben UK Ltd., Suffolk, UK). Samples weremounted on aluminum stabs using tissue freezing medium (Ted Pella, Inc.,Calif., USA), and cooled to −24° C. Images were captured at 15 kVaccelerating voltage.

Contact angle: The contact angle of the ECM electrospun fibrousscaffolds was determined by disposing a droplet of near 1 mm in diameteronto the surface. A static image was then captured with an Artcam 130MIBW camera (Artray Co. Ltd., Tokyo, Japan). The contact angle was thencalculated using a special procedure developed in MatLab R2014a software(Mathworks, Natick, Mass., USA).

FTIR: Fourier transform infrared spectroscopy (FTIR) spectra of theelectrospun ECM scaffolds and decellularized ECM were recorded using aThermo 6700 FUR instrument, equipped with a Smart iTR Attenuated TotalReflectance (ATR) diamond plate, in the wave-number range of 500-3500cm-1 (64 scans at a resolution of 4 cm-1, n=4). Data were evaluatedusing OMNICTM series software (version 8, Thermoscientific). Secondarystructure of proteins was determined by Fourier deconvolution of theiramid I band.

TGA: Thermal gravimetric analysis (TGA) data were obtained using aTGA-Q5000 system (TA instruments, USA). Samples were heated from RT at arate of 20° C. min⁻¹ under nitrogen atmosphere to a final temperature of600° C. Data were analyzed using TA Universal Analysis Software (TAInstruments, New Castle, Del., USA).

Electrospun ECM scaffold composition: An analysis of the proteincomposition was performed at the Proteomics Center, Technion—IsraelInstitute of Technology. Samples were digested by trypsin and theresulting peptides were analyzed by LC-MS/MS. After which, the peptidemix was fractionated by HPLC and electro-sprayed onto an ion-trap massspectrometer, in order to determine the proteins' mass. The peptideswere further fragmented by collision induced dissociation and analyzedagain, for additional analysis and identification. Peptides wereanalyzed and identified using Proteome Discoverer™ software(Thermo-Scientific) against the porcine part of the UniProt database.

Mechanical testing: Tensile properties of the electrospun 3D fibrousscaffolds were determined using a dynamic mechanical analyzer (DMA) Q800(TA Instruments, New Castle, Del., USA) at ambient conditions. Scaffoldswere cut into 4.3±0.2 mm by 6.7±0.3 mm rectangular specimens, mountedvertically between two mechanical gripping units, in a physiologicalbuffer, An extension rate of 2% min⁻¹ was then applied until the yieldpoint. Stress-strain curves were recorded using TA Universal Analysis2000 v. 4.5 A software (TA instruments) and the ultimate stress, strainand ultimate stress, and Young's modulus at 2% strain were extrapolatedfrom the graph.

Cell culturing on electrospun (ES)-pcECM scaffolds: Scaffolds were cutinto circles (D=0.18 cm), placed in 96-well plates, and sterilized usingultra-violet light. Culture medium was added and plates were then movedinto a humidified incubator (37° C., 5% CO₂). One hour later, the mediumwas washed, replaced with a fresh one and the plates were returned tothe incubator overnight for wetting. The following day, cells wereseeded.

Human bone marrow mesenchymal stem cells (hMSCs, Lonza, Basel,Switzerland) were seeded (10,000 per scaffold) and cultured for 1 month.hMSCs were cultured in αMEM, supplemented with FCS (10%), pen-strep(1%), fimgizone (0.4%), and basic fibroblast growth factor (5 ng mL⁻¹).The medium was replaced every second day. Cell viability was evaluatedusing the AlamarBlue™ reagent (AbD Serotec, Kidlington, UK), accordingto the manufacturer's protocol. hiPSCs were seeded (30,000 per scaffold)and cultured for 1 month. The hiPSCs were cultured in mTeSR™ BasalMedium and supplemented with mTeSR™ 1 5× supplement. The medium wasreplaced every day. Neonatal cardiomyocytes were isolated from 24-hr-oldWister rats. Excised hearts were minced, and the cardiac cells weredissociated by gentle agitation in 200U mL⁻¹ RDB in PBS-G (PBS,pen-strep, 0.1% D-glucose) at 37° c for 10 min. Cell suspensions werecentrifuged at 1000 rpm for 5 min, suspended in F-10 nutrient mixturesupplemented with 5% fetal bovine serum, 5% DHS, 1% pen-strep and 0.4%fungizone and 1 mM CaCl₂. Cell suspensions was pre-plated in culturedishes and incubated for 1 hr to allow adherence of the fibroblastcells. The non-attached myocyte-enriched cell suspension was collected,centriffiged as before, and re-suspended in culture medium. Neonatalcardiomyocytes were seeded on scaffolds (200,000 cells/scaffold, n=12)and cultured for 3 weeks in the supplemented F-10 nutrient mixture. Themedium was changed every second day.

Lightsheet Fluorescent Microscopy (LSFM): To study the morphology ofcells cultured on electrospun ECM scaffolds, cells were stained with FDA(for viable cells), and with Hoechst 33258 (for DNA). Scaffolds werethen embedded within low-melting to Agarose gel (Bio-Rad, Haifa, Israel)and inserted within a 1 mL syringe. The sample was then observed usinglight sheet fluorescence microscopy (LSFM) using a Lightsheet Z.1(Zeiss), equipped with dual sided beam illumination with two alignedobjectives for complete, high resolution 3D imaging, two cameras, and afull incubation compartment for live samples.

Histology: Cell-seeded scaffolds were fixed in PEA (4%) for 20 min,washed in PBS and frozen in Tissue-Tek™ OCT compound, cross-sectionedinto slices (10 μm) on glass slides and stained. Slides were fixed incold WWI (4° C.) for 20 min prior to staining. After fixation_(;) slideswere washed in DDW 3-5 times to remove all the OCT compound, and stainedwith hematoxylin and eosin (H&E). Immunofluorescent staining for cardiacmarkers was performed using cTn1, sarcomeric alpha-actinin, andconnexin-43 primary antibodies, according to the manufacturers'protocol. Slides were visualized by inverted phase-contrast microscopy(Eclipse TE2000-E, Nikon Inc.).

Ca²⁺ imaging: Cells were loaded with 5 mM of fluo-4 fluorescent Ca²⁺indicator (Molecular Probes) in the presence of Plutonic F-127(Molecular Probes) at a dilution of 2:1 to allow the recording ofintracellular Ca²⁺-transients (whole-cell [Ca²⁺] transients). Forpacing, scaffolds were plated on a 35-mm optical plate (Matek) withfield simulation electrodes (RC-37FS; Warner Instruments), and pacedusing a stimulus isolation unit (SIU-102, Warner Instruments), byapplying 5 ms-suprathreshold bipolar stimulation pulses up to 50 mA.Intracellular Ca²⁺-transients were recorded using a Zeiss laser-scanningconfocal imaging system (Flux-view; Olympus) mounted on an uprightBX151WI Olympus microscope equipped with a X60 water objective. Datawere analyzed utilizing MatLab-based custom-written software.

MSCs remodeling of peECM-based scaffolds; The expression of ECMremodeling-related genes by cells seeded on the pcECM-based scaffoldswas quantitatively studied for 21 days, by real-time RT-PCR. Thefollowing genes were studied: Collagen I (α1 chain), collagen III (α1chain), matrix metalloproteinase 2 (MMP2), and type 1 tissue inhibitorof metalloproteinases (TIMP1). Total RNA was isolated from the seededcells at different time points using Tri-reagent (Sigma-Aldrich)according to the manufacturer's instructions, and reverse-transcribed ina PTC-200 PCR cycler using a Verso™ cDNA kit (Thermo-Scientific).Primers were designed to specifically amplify genes' cDNA as follows:

-   5′-TACAGCGTCACTGTCGATGGC-3′ (SEQ ID NO: 5) and    5′-TCAATCACTGTCTTGCCCCAG-3′ (SEQ ID NO: 6) for collagen Iα1.-   5′-AATTTGGTGTGGACGTTGGC-3′ (SEQ ID NO: 7) and    5′-TTGICGGICACTTGCACTGG-3′ (SEQ ID NO: 8) for collagen III α1.-   5′-TTGACGGTAAGGACGGACTC-3′ (SEQ ID NO: 9) and    5′-ACTTGCAGTACTCCCCATCG-3′ (SEQ ID NO: 10)for MMP2.-   5′-TACTTCCACAGGTCCCACAA-3 (SEQ ID NO: 11) and    5′-ATTCCTCACAGCCAACAGTG-3′ (SEQ ID NO: 12) for TIMP1.-   5′-CAACAGCGACACCCACTCCT-3′ (SEQ ID NO: 13) and    5′-CACCCTGTTGCTGTAGCCAAA-3′ (SEQ ID NO: 14) for glyceraldehyde    3-phosphate dehydrogenase (GAPDH) as an intrinsic housekeeping gene    control. Reactions were run on the StepOnePlus system, and analyzed    using StepOne software v. 2.2.2 (Applied Biosystems).

In vitro immunogenicity assay: Macrophage stimulation was used toevaluate the immunogenicity of the ECM electrospun scaffolds in-vitro.Both levels of secreted nitric oxide (NO) and expression ofpro-inflammatory cytokines were measured. RAW cell line (TIB-71™; ATCCManassas, Va., USA) was seeded in 24-well cell culture plates andcultured in 1 mL high-glucose DMEM supplemented with 10% FCS, 1%Pen-Strep, and 0.4% Fungazone. The following day post seeding, mediumwas replaced with a low-serum medium (2% FCS). When cells reached 70%confluency, cells were exposed to the following: electrospun ECMscaffolds (1 mg), PLGA as a negative control (1 mg), or LPS as apositive control (1 μg/mL). After a 16 h incubation, secreted NO levelswere measured as the free stable nitrite form (NO₂) in the medium, usingthe conventional Griess Reagent Assay (reference). Untreated cells alsoserved as a negative control (basal NO secretion). Additionally, totalRNA was isolated from the seeded RAW macrophages using Tri-reagent(Sigma Aldrich) according to the manufacturer's protocol, andreverse-transcribed in a PTC-200 PCR cycler using a Verso™ cDNA kit(Thermo-Scientific, Waltham, Mass., USA). The isolated. RNA was thenused for real-time (RT-) PCR analyses in order to quantify theexpression of the pro-inflammatory cytokines TNF-α and IL1-β with thefollowing specific primers.

(SEQ ID NO: 1) 5′-GCCTCCCTCTCATCAGTTCT-3′ and (SEQ ID NO: 2)5′-TGGTGGTTTGCTACGACGTG-3′ for TNF-α (SEQ ID NO: 3)5′-AGGATGAGGACATGAGCACC-3′ and (SEQ ID NO: 4) 5′-ATGGGAACGTCACACACCAG-3′for IL-1β

In vivo immunogenicity study: Immunogenicity experiments were conductedin accordance with the Israeli Animal Welfare (Protection andExperimentation) Law, after obtaining the permission of the Technion'sAnimal Care Committee. The immunogenic potential of ECM electrospunfibrous scaffolds was additionally evaluated in vivo throughsubcutaneous implantation. Mice were split into two groups; one groupreceived the electrospun ECM scaffold, and the other received theelectrospun PLGA scaffold as a negative control. Each group was splitinto three time points (1, 2, and 4 weeks). There were five mice pergroup per time point.

On the day of the surgery, six-week old C57BL mice (Pharma Medis Ltd,Holon, Israel) were anesthetized with a 300 μL peritoneal injection ofketamine (100 mg/kg) (Vetoquinol, Lure, France) and xylasine (5 mg/kg)(Phibro Israel, Beit Shemesh, Israel), shaved on the right flank, andsubsequently given 150 μL buprenorphine (0.05 mg/kg) (Vet Market,Shoham, Israel) via subcutaneous injection for pain. During theprocedure, an isofluorane/oxygen mixture (2-3% isofluorane) wasadministered via gas mask for maintenance. Mice were placed on a heatingpad (37 ° C.) and a subcutaneous incision was made on the shaven rightflank through which the respective scaffold was inserted. Incisions werestitched and mice were placed into an oxygen-rich, temperaturecontrolled (X ° C.) environment. Mice were routinely monitored, andsacrificed at the respective time points. Blood samples were takenpost-mortem from the heart and/or femoral artery for complete bloodcounts (CBC), inguinal lymph nodes, scaffolds were analyzed. Lymph nodeswere homogenized, the mRNA was extracted and reversed transcribed, andproinflammatory cytokines (TNFα and IL-1β levels normalized to GAPDH)were quantified as previously described.

RESULTS

ECM Dissolution and Electrospinning

ECM was obtained from slices of porcine tissues that were decellularizedas described in WO 2006095342A2 and in the Materials and methods sectionherein above, which avoided the use of SDS and allowed for improvedpreservation of ECM biological activity. ECM were frozen in liquidnitrogen, ground in a cryogenic tissue grinder, and lyophilized. Theresulting powder was used for dissolution.

Lyophilized ECM was dissolved in HFIP to a concentration of 0.05 g/mLand pcECM solution was added with 0.1 mass (4) PEO. Without PEO, pcECMelectrospinning resulted in a combination of fibers and spray (FIG. 1A);while with PEO, a network of homogeneous fibers were achieved (FIG. 1B).The PEO was easily removed from the matrix through washes in awater-based medium, and the pcECM fibers remained intact.

Scaffold morphology and characterization: HR-SEM images were used toexamine the morphology and diameter of the fibers within ECM electrospunfibrous scaffolds (FIGS. 2A-D, FIGS. 3A-B). At lower magnifications(0.2-1 kX), the fibers of the electrospun pcECM scaffold appearedrandomly dispersed, with minimal non-fiber aggregates, which weresuspected to be pieces of collagen that were not fully dissolved (FIGS.2A-B). At higher magnitudes (5 kX), two types of fibers were apparent,one quite thick and the other thin. At even higher magnification, (10kX) the morphology of the porous structure of the individual fibers wasvisible (FIG. 2D). According to image.1 analyses, the diameter of thelarger fibers was approximately 734±228 nm, ranging from 300 to 1500 nm,exhibiting a normal distribution (FIG. 5), consistent with naturalECM.¹⁰ The diameter of the thinner fibers was approximately 43±7.9 nm,which still falls within the range of the fiber diameter seen in naturalECM. Pore size of the electrospun pcECM scaffold ranged between 1-30μm², with an average of 7.6±1.7 μm² (FIG. 6).

The contact angle of the ECM electrospun fibrous scaffolds was observedby dropping a 1 mm droplet of DDW on the surface, and capturing a staticimage. Analysis was performed using a home-designed MatLab program. Thewater contact angle of electrospun pcECM and ppECM scaffolds was96.33±2.12° or 74.3 respectively. After less than 5 minutes, the contactangle of electrospun pcECM decreased to 60° (FIG. 7A-B and FIG. 8).

ECM electrospun fibrous scaffolds composition: Since the proteincomposition is a major factor contributing to the biological activityand mechanical properties of the scaffold, possible changes in proteincomposition were first evaluated by comparing FTIR spectra obtained fromdecellularized pcECM to those of the ES-pcECM scaffold (FIG. 9A-B). Bothmaterials exhibited amide vibrations, near and above 3000 cm⁻¹,characteristic of peptide groups, and vibrations between 500-1700 cm⁻¹,characteristic of amino acid side chains, with no significantdifferences between decellularized pcECM and ES-pcECM scaffold.Moreover, similar percentages of each secondary structure weredetermined by Fourier deconvolution. Proteomic mass spectrometryanalyses were performed to ensure that proteins were not lost during thedissolution or electrospinning process. Since collagen is the mostabundant protein in ECM, and provides the necessary tensile strength andviscoelasticity, the collagen content was inspected thoroughly (Table 1,Table 2, and FIG. 3),

TABLE 1 Accession # Gene Level of abundance F1SFA7 Collagen alpha-2(I)Level 1 F1RYI8 Collagen alpha-1(III) Most abundant proteins F1RT61Collagen alpha-1(I) I3LJX2 similar to collagen alpha-1(I)* F1RLL9(Fragment) Collagen alpha-2(IV) Level 2 F1S021 (Fragment) Collagenalpha-1(V) >3 fold decrease from I3LSV6 (Fragment) Collagen alpha-1(II)level 1 Q59IP2 Procollagen alpha 2(V) I3LQ84 Collagen alpha-2(VI)**Level 3 I3LUR7 Collagen alpha-3(VI) >20 fold decrease from F1RLM1(Fragment) Collagen alpha-1(IV) level 1 I3LS72 Collagen alpha-1(VI) >3fold decrease from F1S3G7 (Fragment) Collagen alpha-3(V) level 2 F1SKX7Collagen alpha-1(VIII) Level 4 D5KRL1 Collagen alpha-1(XXI) >100 folddecrease from level 1 >60 fold decrease from level 2 >8 fold decreasefrom level 3

Table 2 presents the collagenous composition of electrospun porcinepancreatic ECM (ppECM) scaffold as revealed in proteomic analysis.

Gene Name Score Coverage COL1A2 496.16 52.2 COL3A1 190.58 21.7 COL1A180.31 66.5 COL2A1 39.48 3.2 COL5A1 25.32 3.1 COL5A2 24.31 8.9 COL5A34.65 3.4

All collagen types were grouped by their level of abundance as accordingto their mass spectrometry results; level 1 signifying the most abundantcollagen. When a significant fold decrease was detected, the collagenwas placed in the subsequent level. For example, all the collagen inlevel 2 had larger than a 3 fold decrease in their level of abundancefrom all the collagen in level 1. Level 3 collagen decreased inabundance more than 2.0 fold from level 1 and 3 fold from level 2, whilelevel 4 collagen decreased more than 100 fold from level 1, 60 fold fromlevel 2, and 8 fold from level 3. These groupings allowed for a morein-depth analysis of the collagen composition of the pcECM electrospunscaffold, Level 1 contains only collagen types I and III, which isexpected, since collagen types I and III, usually found together, arethe most plentiful collagen within cardiac ECM tissue, (>90%).¹⁵ Theyare fibril, interstitial collagen types that maintain tissue structureand support cardiomyocytes.

Levels 2 and 3 contain the less abundant fibrillar forming collagens (IIand V), and network forming collagen that is present in basementmembranes (IV). Level 3 also contains collagen type VI, which provides amicrofilament network that organizes the fibrillary collagens andanchors them to the basement membranes. Collagen alpha-2 (VI) was theonly collagen that exhibited a significant fold decrease in the pcECMelectrospun scaffold from the decellularized pcECM. In general, it wasdetermined that, with the exception of collagen alpha-2 (VI), there wasno significant fold difference in the collagen content between thedecellularized pcECM and the pcECM electrospun scaffold,

TGA analyses (FIG. 10), compared the characteristic thermal degradationof the scaffold to that of decellularized pcECM. The onset ofdecomposition (T_(onset)) was similar for both materials: 259.4° C.(scaffold) and 259.6° C. (decellularized pcECM), signifying that theproduction process of ES-pcECM scaffolds, and particularly thedissolution of pcECM in HFIP, had not degraded the collagen.

Self assembly of electrospun peECM: Upon wetting the ES-pcECM scaffold,the electrospun fibers underwent self-assembly, consequently obviatingthe need for a synthetic cross-linking agent (FIG. 11). The extent ofself-assembly varied according to the initial wetting temperature aswell as the subsequent incubation temperature. The largest degree ofself-assembly was obtained when ES-pcECM scaffolds were maintained at37° C., where the ES-pcECM self-assembly produced an isotropic, porous,ordered structure that most clearly and uniquely resembled themicrostructure of native pcECM. ES-pcECM scaffolds maintained at 24° C.showed a lower degree of self-assembly, and those maintained at 4° C.showed minimal self-assembly, and anisotropic structures withelectrospun fibers still very apparent. Increasing the incubationtemperature post initial wetting (from 4° C. and 24° C.) to 37° C.increased their self-assembly; however, complete self-assembly was notachieved, and electrospun fibers were still present to some extent.These studies provide a wealth of data regarding the abilities of theES-pcECM scaffold, which had not been observed with the electrospinningof other natural polymers.

Mechanical properties of the electrospun pcECM scaffold: The mechanicalproperties of an engineered scaffold are particularly critical whenaddressing cardiac regeneration. The mechanical properties of theelectrospun pcECM scaffold were compared to those of native tissue in aphysiological solution (FIG. 12 A-D). The stress and strain at maximumstress were similar in the electrospun pcECM scaffold and native cardiactissue (p>0.05). The Young's modulus (E) of the scaffolds (029±0.007MPa) was also comparable to that of native tissue (0.22±0.007), thoughstatistically to different (p<0.05). Such findings confirmed thatself-assembly of electrospun pcECM scaffold is sufficient to obtain themechanical properties desired for cardiac regeneration with no need fora synthetic cross-linking agent.

Culturing hMSCs on pcECM electrospun fibrous scaffolds: The ability forcells to adhere to and proliferate on the pcECM electrospun fibrousscaffold was assessed in terms of the viability of human mesenchymalstem cells (hMSCs). Viability studies confirmed that the pcECMelectrospun scaffold supported the hMSCs, which remained at an averagedensity of 80,000 cells per scaffold for up to 4 weeks (FIG. 13).

hMSCs portrayed normal elongated morphology and scaffold penetration(FIGS. 14A-D). The presence of adherent, viable hMSCs after 1 month ofculture on the scaffold was also supported by light sheet fluorescencemicroscopy (LSFM) analysis (FIGS. 15A-B). Hematoxylin and eosin (II&E)histological analysis demonstrated that the cells had integrated withinthe matrix (FIG. 15C).

The seeded hMSCs' ability to remodel the electrospun pcECM scaffold wasevaluated by analyzing their expression of ECM-remodeling related genes(FIG. 16). In both native ECM and the electrospun ECM scaffold, collagenI expression increased 3, 7, and 14 days post seeding in 10-20 foldscompare to the basal level. This elevated expression decreased by day21. Maximal increase of collagen III expression was observed in day 3for the native ECM (13 fold) and day 21 for the electrospun ECM scaffold(18 fold). TIMP1 (collagenase inhibitor), maximal expression wasobserved at day 7 for the electrospun scaffold (25 fold) and day 14 forthe native ECM (7 fold). MMP2, an indicator collagenase, increased atday 7, and reached maximal expression at day 14 on both native ECM andthe electrospun ECM scaffold.

Human induced pluripotent stem cells (hiPSC) were seeded on electrospunECM scaffolds, exhibiting a confluent matrix after three weeks inculture (FIG. 17), with impressive viability levels demonstrated throughthe small portion of dead cells (propidium iodide, pink). CryoSEM imagesrevealed adherent hiPSCs in their regular cluster morphology (FIG.18A-C), and H&E analysis showed their integration within the matrix(FIG. 18D).

Neonatal rat cardiomyocytes (rCM) seeded on the electrospun pcECMscaffolds were assessed after 21 days using SEM and LSFM (FIGS. 19A-B),and revealed normal spherical morphology. H&E staining demonstrated thecells' integration within the matrix (FIG. 19C). The seeded rCM werepositively stained for the typical functional cardiac proteins cardiactroponin I (cTn1), sarcomeric alpha-actinin and connexin-43 (FIG.20A-C), indicating contractile functioning as well as cell—cell couplingthrough functional gap junctions. This contractile function was alsodemonstrated through the spontaneous beating of the seeded scaffolds,initiated less than 2 days post seeding. After approximately two weeks,the synchronically beating scaffold was analyzed using Ca²⁺ imaging torecord beating and to test electrical coupling. The action potentialproperties evaluated during pacing (using field stimuli) at differentrates (FIG. 21) further confirmed that the electrospun pcECM scaffoldcan support the cells' intact synchronized electrical activity.

The cultivation of an additional type of cells—hiPSC-derivedcardiomyocytes (hiPSC-CM)—on electrospun pcECM scaffold revealed highviability levels 14 days and 3 weeks post seeding (FIGS. 22, FIG. 23).Contractile function was also demonstrated through the spontaneousbeating of the seeded scaffolds, initiated less than 24 hr post seeding.After approximately two weeks, the synchronically beating scaffolds wasanalyzed using Ca²⁺ imaging to test electrical coupling. As seen in FIG.24, whole-cell [Ca²⁺] transients during pacing (using field stimuli) at1 Hz rates was achieved, displaying as a line-scan tracing and furtherconfirming that the electrospun pcECM scaffold can support the cells'intact synchronized electrical activity.

Immunogenicity Studies

Immunogenicity of pcECM electrospun scaffolds was examined both in vitroand in vivo. RAW macrophages were stimulated with crushed pcECMscaffolds, PLGA (negative control), lipopolysaccharide (LPS-positivecontrol). Non-stimulated cells were also used as a negative control.After a 16 h incubation period with the stimulated materials, the levelof secreted nitric oxide (NO) was evaluated (FIG. 25A). There was nosignificant change observed in the level of secreted NO between thepcECM scaffolds, PLGA, and non-stimulated cells (P>0,05). However, thelevel of secreted NO was significantly higher (P<0.01) for cellsstimulated with LPS as compared to to pcECM scaffolds and both negativecontrols, which demonstrated that LPS was a successful positive control.Pro-inflammatory cytokine expression of TNF-α and IL1-β for the RAWmacrophages were also evaluated 16 h post stimulation with real-time(RT-) PCR analysis (FIGS. 2513, C). Similar results to the NO excretionwere obtained. The expression of the pro-inflammatory cytokine TNF-αshowed a 1.66±0.64 fold increase for RAW macrophages stimulated withpcECM scaffolds, while there exhibited at 0.73±0.65- and a67.5±32.6-fold increase for RAW macrophages stimulated with PLGA andLPS, respectively it1 respect to non-stimulated cells). Only the foldincrease of the TNF-α expression exhibited by RAW macrophages stimulatedwith LPS can be considered highly significant (P<0.01). The expressionof the pro-inflammatory cytokine IL1-β showed a 2.69±1.79 fold increasefor RAW macrophages stimulated with pcECM scaffolds, while thereexhibited at 0.25±0.26-fold and a 18,124±14,581-fold increase for RAWmacrophages stimulated with PLGA and LPS, respectively (with respect tonon-stimulated cells). Only the fold increase of the IL1-β expressionexhibited by RAW macrophages stimulated with LPS can be consideredhighly significant (P<0.01).

The immunogenicity of pcECM electrospun fibrous scaffolds wasadditionally evaluated in vivo through subcutaneous implantation. Micewere split into two groups; one group received the pcECM scaffold, andthe other received electrospun PLGA scaffolds as a negative control.One, two and four weeks following implantation the mice were sacrificed.Lymph node examination revealed no swelling or irritation in all testedgroups. In addition, the expression of pro-inflammatory cytokines TNF-αand IL1-β in the lymph nodes revealed no significant difference betweenthe ES-pcECM scaffold groups and the ES-PLGA scaffold groups of the sameweek (FIGS. 26A-B). Complete blood counts (CBC) revealed no increase inthe levels of white blood cells (WBCs), red blood cells (RBCs),hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), MCH concentration (MCHC), neutrophils, and lymphocytesat the ES-pcECM scaffold treatment group compared to the PLGA negativecontrol group for all time points (FIG. 27 A-I).

Overall, these immunogenicity studies demonstrated that pcECMelectrospun fibrous scaffolds, although taken from a porcine source andsolubilized in a hazardous organic solvent, are non-immunogenic. Thus,they are candidates for use a biocompatible cardiac scaffolds, forrepairing damaged tissue.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

REFERENCES

1. Badylak, S. E. The extracellular matrix as a scaffold for tissuereconstruction. Seminars in Cell & Developmental Biology 13, 377-383(2002),

2. Uriel, S. et al. Extraction and assembly of tissue-derived gels forcell culture and tissue engineering. Tissue Engineering Part C: Methods15, 309-321 (2008),

3. Ghorani, B. & Tucker, N. Fundamentals of electrospinnin,s7, as anovel delivery vehicle for bioactive compounds in food nanotechnology.Food Hydrocolloids 51, 227-240 (2015).

4. Yu, J. H., Fridrikh, S. V. & Rutledge, G. C. The role of elasticityin the formation of electrospun fibers. Polymer 47, 4789-4797 (2006).

5. Dong, B., Arnoult, O., Smith, M. E. & Wnek, G. E. Electrospinning ofCollagen Nanofiber Scaffolds from Benign Solvents. Macromolecular RapidCommunications 30, 539-542 (2009).

6. Punnoose, A. M., Elamparithi, A. & Kuruvilla, S. Electrospun Type 1Collagen Matrices Using a Novel Benign Solvent for Cardiac TissueEngineering. J Cell Physiol (2015).

7. Nieuwland, M. et al. Food-grade electrospinning of proteins.Innovative Food Science & Emerging Technologies 20, 269-275 (2013).

8. Burck, J. et al. Resemblance of electrospun collagen nanofibers totheir native structure. Langmuir: the ACS journal of surfaces andcolloids 29, 1562-1572 (2013).

9. Heydarkhan-Hagvall, S. et al. Three-dimensional electrospun ECM-basedhybrid scaffolds for cardiovascular tissue engineering. Biomaterials 29,2907-2914 (2008).

10. Barnes, C. P., Sell, S. A., Boland, E. D., Simpson, D. G. & Bowfin,G. L. Nanofiber technology: Designing the next generation of tissueengineering scaffolds. Advanced Drug Delivery Reviews 59, 1413-1433(2007).

11. Jugdutt, B. I. Remodeling of the myocardium and potential targets inthe collagen degradation and synthesis pathways. Current DrugTargets-Cardiovascular & Haematological Disorders 3, 1-30 (2003).

12. Gibson, M. et al. Tissue Extracellular Matrix NanoparticlePresentation in Electrospun Nanofibers. Biomed Research International(2014).

13. Sethuraman, A., Han, M., Kane, R. S. &. Belfort, G. Effect ofsurface wettability on the adhesion of proteins. Langmuir 20, 7779-7788(2004).

14. Kitsara, M. et al. Fabrication of cardiac patch by using electrospuncollagen fibers. Microelectronic Engineering 144, 46-50 (2015).

15. Corda, S., Samuel, J. L. & Rappaport, L. Extracellular matrix andgrowth factors during heart growth. Heart failure reviews 5, 119-130(2000).

16. Kwak, H.-B. Aging, exercise, and extracellular matrix in the heart.Journal of exercise rehabilitation 9, 338-347 (2013).

17. Luther, D. J. et al. Absence of Type VI Collagen ParadoxicallyImproves Cardiac Function, Structure, and Remodeling After MyocardialInfarction. Circulation Research 110, 851-U125 (2012).

18. Silva, G. V. et al. Mesenchymal stem cells differentiate into anendothelial phenotype, enhance vascular density, and improve heartfunction in a canine chronic ischemia model. Circulation 111, 150-156(2005).

19. Fukuda., K. Development of regenerative cardiomyocytes frommesenchymal stem cells for cardiovascular tissue engineering. Artificialorgans 25, 187-193 (2001).

20. McAnulty, R. J. Fibroblasts and myofibroblasts: their source,function and role in disease. The international journal of biochemistry& cell biology 39, 666-671 (2007).

21. Eitan, Y., Sang, U., Dahan, N. & Machluf, M. Acellular cardiacextracellular matrix as a scaffold for tissue engineering: in vitro cellsupport, remodeling, and biocompatibility. Tissue Eng Part C Methods 16,671-683 (2010).

1. A method of generating a scaffold comprising: (a) homogenizingdecellularized extracellular matrix (ECM) in an organic solvent togenerate a homogenate of decellularized ECM; (b) electrospinning saidhomogenate onto a solid surface thereby generating the scaffold. 2.(canceled)
 3. The method of claim 1, further comprising decellularizinga tissue of a subject prior to generate said decellularized ECM prior tostep (a).
 4. The method of claim 1, further comprising contacting saidsolution of decellularized ECM with a polymer so as to increase theviscoelasticity of said solution following step (a) and prior to step(b).
 5. (canceled)
 6. The method of claim 1, further comprisingfiltering said homogenate of decellularized ECM prior to said electrospinning.
 7. The method of claim 1, wherein said organic solvent isselected from the group consisting of acetone, N,N-dimethylformamide(DMF), diethylformamide, chloroform, methylethylketone, acetic acid,formic acid, ethanol, 1,1,1,3,3,3 -hexa fluoro-2-propanol (HFIP),tetrafluoroethanol, dichloromethane (DCM), tetrahydrofuran (THF),trifluoroacetic acid (TFA), camphorsulfonic acid, dimethyl acetamide,isopropyl alcohol (IPA) and mixtures thereof.
 8. The method of claim 1,wherein said organic solvent is HFIP.
 9. The method of claim 4, whereinsaid polymer is a biocompatible polymer.
 10. The method of claim 4,wherein said polymer is a hydrophilic polymer.
 11. The method of claim4, wherein said polymer is a synthetic polymer.
 12. The method of claim11, wherein said synthetic polymer is selected from the group consistingof poly(D,L-lactide) (PLA), poly(urethanes), poly(siloxanes),poly(silicones), poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxyethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methylmethacrylate), poly(vinyl alcohol) (PVA), poly(acrylic acid), poly(vinylacetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethyleneglycol), poly(methacrylic acid), polyglycolic acids (PGA),poly(lactide-co-glycolides) (PLGA), nylons, polyamides, polyanhydrides,poly(ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly(vinylacetate), polyvinylhydroxide, poly(ethylene oxide) (PEO),polyorthoesters and mixtures thereof.
 13. The method of claim 4, whereinsaid synthetic polymer is PEO.
 14. The method of claim 13, wherein theamount of said PEO in said solution is between 0.05-1% mass.
 15. Themethod of claim 1, wherein said decellularized ECM is derived from anorgan selected from the group consisting of heart and pancreas.
 16. Themethod of claim 1, wherein said decellularized ECM is derived fromporcine tissue.
 17. The method of claim 1, further comprising removingsaid polymer following said electrospinning.
 18. A scaffold generatedaccording to the method of claim
 1. 19. A scaffold comprisingelectrospun decellularized ECM of an organ, wherein said decellularizedECM has a similar protein composition to native ECM of said organ.20-21. (canceled)
 22. The scaffold of claim 19, wherein said organ is aheart or a pancreas.
 23. The scaffold of claim 9, wherein said organ isa human organ or a porcine organ.
 24. The scaffold of claim 19, whereinsaid decellularized ECM comprises collagen type I and collagen type III.25. The scaffold of claim 19, wherein said decellularized ECM is devoidof collagen type VI.
 26. The scaffold of claim 19, wherein the diameterof fibers of the scaffold are between 100-2000 nm.
 27. The scaffold ofclaim 19, wherein the diameter of fibers of the scaffold are between 300to 1500 nm.
 28. The scaffold of claim 19, wherein, when hydrated, thefibers of the scaffold have a similar organization to native ECM of saidorgan.
 29. The scaffold of claim 19, being devoid of a syntheticpolymer.
 30. (canceled)
 31. A method of treating a medical conditionwhich may benefit from cell transplantation in a subject in needthereof, comprising transplanting the scaffold of claim 19 into thesubject, thereby treating the medical condition.
 32. The method of claim31, wherein the scaffold has been pre-seeded with cells.
 33. The methodof claim 31, wherein the medical condition is a cardiac disease.
 34. Themethod of claim 33, wherein the medical condition is Diabetes. 35.(canceled)